Cellular differentiation of multicellular organisms is controlled by hormones and polypeptide growth factors. These diffusable molecules allow cells to communicate with each other, to act in concert to form tissues and organs, and to repair and regenerate damaged tissue. Examples of hormones and growth factors include the steroid hormones, parathyroid hormone, follicle stimulating hormone, the interferons, the interleukins, platelet derived growth factor, epidermal growth factor, and granulocyte-macrophage colony stimulating factor, among others.
Hormones and growth factors influence cellular metabolism by binding to receptor proteins. Certain receptors are integral membrane proteins that bind with the hormone or growth factor outside the cell, and that are linked to signaling pathways within the cell, such as second messenger systems. Other classes of receptors are soluble intracellular molecules.
Wnt proteins are emerging as one of the pre-eminent families of signaling molecules in animal development. To date, murine Wnt genes include Wnt-1, Wnt-2, Wnt-2B/13, Wnt-3, Wnt-3A, Wnt-4, Wnt-5A, Wnt-5B, Wnt-6, Wnt-7A, Wnt-7B, Wnt-8A, Wnt-8B, Wnt-10A, Wnt-10B, Wnt-11, and Wnt-15, while the following human Wnt genes have been described: Wnt-1, Wnt-2, Wnt-2B/13, Wnt-3, Wnt-4, Wnt-5A, Wnt-7A, Wnt-8A, Wnt-8B, Wnt-10B, Wnt-11, Wnt-14, and Wnt-15. See, for example, Nusse and Varmus, Cell 31:99 (1982), van Ooyen et al., EMBO J. 4:2905 (1985), Wainwright et al., EMBO J. 7:1743 (1988), McMahon and McMahon, Development 107:643 (1989), Gavin et al., Genes Dev. 4:2319 (1990), Roelink et al., Proc. Nat'l Acad. Sci. USA 87:4519 (1990), Roelink and Nusse, Genes Dev. 5:381 (1991), Clark et al., Genomics 18:249 (1993), Roelink et al., Genomics 17:790 (1993), Adamson et al., Genomics 24:9 (1994), Huguet et al., Cancer Res. 54:2615 (1994), Bouillet, Mech. Dev. 58:141 (1996), Ikegawa et al., Cytogenet. Cell Genet. 74:149 (1996), Katoh et al., Oncogene 13:873 (1996), Lako et al., Genomics 35:386 (1996), Wang and Shackleford, Oncogene 13:1537 (1996), Bergstein, Genomics 46:450 (1997), Bui et al., Oncogene 14:1249 (1997), and Grove et al., Development 125:2315 (1998).
Wnt genes typically encode secreted glycoproteins having 350–400 amino acids, and the proteins often include a conserved pattern of 23–24 cysteine residues in addition to other invariant residues (Cadigan and Nusse, Genes & Dev. 11:3286 (1997)). Following cellular secretion, Wnt proteins are believed to reside mainly in the extracellular matrix or to associated with the cellular surface.
According to the classical Wnt signaling pathway model, Wnt proteins induce gene expression by de-repressing a signal pathway via a so-called “Frizzled” transmembrane receptor (see, for example, Brown and Moon, Curr. Opin. Cell Biol. 10:182 (1998)). In the absence of Wnt, glycogen synthase kinase-3β activity results in the degradation of the free cytosolic pool of β-catenin. The association of cognate Wnt proteins and Frizzled receptors leads to the activation of a signaling pathway. The most proximal intracellular component of this pathway is the Disheveled protein, which becomes phosphorylated and inhibits glycogen synthase kinase-3β. Consequently, the pool of intracellular β-catenin increases, and β-catenin can interact with members of the lymphoid enhancer/T cell factor (LEF/TCF) family of architectural transcription factors in the nucleus. These complexes bind consensus LEF/TCF sites in promoters and induce transcription of Wnt-responsive genes.
The Wnt proteins are multipotent, and the proteins are capable of inducing different biological responses in both embryonic and adult contexts (see, for example, Ingham, TlG 12:382 (1996)). This type of broad activity is shared with fibroblast growth factors, transforming growth factors β, and nerve growth factors (Nusse and Varmus, Cell 69:1073 (1992)). When over-expressed, Wnt proteins can promote tumor formation (Erdreich-Epstein and Shackleford, Growth Factors 15:149 (1998)). Knock-out mutations in mice have shown Wnt proteins to be essential for brain development, and the out growth of embryonic primordia for kidney, tail bud and limb bud (McMahon and Bradley, Cell 62:1073 (1990), Thomas and Capecchi, Nature 346:847 (1990), Stark et al., Nature 372:679 (1994), Takada et al., Genes Dev. 8:174 (1994), and Parr and McMahon, Nature 374:350 (1995)).
Several secreted factors inhibit Wnt signaling (see, for example, Finch et al., Proc. Nat'l Acad. Sci. USA 94:6770 (1997); Moon et al., Cell 88:725 (1997); Luyten et al., WO 98/16641); Brown and Moon, Curr. Opin. Cell Biol. 10:182 (1998); Aikawa et al., J. Cell. Sci. 112:3815 (1999)). The Frzb proteins, for example, bind to secreted Wnt proteins and prevent productive interactions between Wnt and Frizzled proteins. These proteins contain a region that is homologous to a putative Wnt-binding domain of Frizzled proteins. Wnt-inhibitory factor-1 is another type of secreted protein, which binds to Wnt proteins and inhibits Wnt signaling (Hsieh et al., Nature 398:431 (1999)). Wnt-inhibitory factor-1 proteins are produced by fish, amphibia, and mammals, indicating the importance of these inhibitory proteins (Hsieh et al., Nature 398:431 (1999)).
Inhibitors of Wnt signaling can be used to block the inducement of tumor formation by inappropriate Wnt expression. Accordingly, a need exists for the provision of new Wnt inhibitory proteins.